Membrane proteins comprise nearly a third of the human genome and over half of all drug targets, yet re- main relatively poorly characterized with less than 1% of all resolved protein structures representing them. Furthermore, the mechanisms of membrane protein development are not well understood, particularly with respect to their folding and membrane insertion. This lack of understanding is especially critical given that the majority of known disease-causing mutations in membrane proteins affect not their function but rather how they develop. To remedy the dearth of structural and developmental information on membrane proteins, the PIs aim to determine new structures and detailed models covering processes relevant to membrane protein folding, insertion, and assembly. By employing advanced computational modeling tools and molecular dynamics simulations, three key deficiencies will be targeted. Through newly resolved structural data and very long (microsecond) simulations, the means by which a nascent membrane protein is oriented and directed from the ribosome, a large molecular machine that synthesizes all proteins, to the translocon, a membrane-bound channel that catalyzes membrane insertion, will be determined. Additionally, the energetics governing insertion of a membrane protein into the membrane as well as its complement, extraction from the membrane, will be quantified and compared with experiments addressing the same insertion and extraction processes. In particular, a close connection to atomic force microscopy experiments will permit the identification and verification of the individual molecular interactions that stabilize a given protein's structure. Finally, the folding propensity of membrane proteins at stages early in their development will be measured, an aspect which is suspected to determine the efficiency of their targeting to the membrane insertion pathway. These measurements will utilize new structures of developmental intermediates solved through the integration of data from multiple experimental sources, including X-ray crystallography and electron microscopy. The unique structures and quantitative models generated through successful achievement of the stated aims will provide the unprecedented atomic-scale detail required both for a complete understanding of membrane protein development and for the design of novel pharmacotherapies. PUBLIC HEALTH RELEVANCE: Membrane proteins make up a third of the human genome and over half of all drug targets, yet the mechanisms of their development remain largely uncharacterized. This project aims to significantly enhance knowledge of these mechanisms at a level of unprecedented detail and realism in order to enable the next generation of knowledge-based design of therapeutic interventions.